Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.
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PMID:CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx. 146 15

CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.
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PMID:Disruption of the CD4-p56lck complex is required for rapid internalization of CD4. 150 68

The compound 12-O-tetradecanoylphorbol-13-acetate (TPA) is extremely toxic to the P13 subclone of the Jurkat human T-cell leukemia line. By selecting for growth in the presence of TPA, we have isolated two TPA-resistant variants of these cells, P13-50 and P13-5/A8. Studies of protein kinase C (PKC) enzyme activity, immunoblot analyses, and assays for PKC mRNAs indicate that both of these variants express lower levels of PKC than do the parental P13 cells. We suggest that this protects them from the toxic effects of TPA. The P13-5/A8 cells are of particular interest because not only are they resistant to TPA toxicity but they actually require TPA for optimal growth. These cells have a more profound decrease in PKC expression that do P13-50 cells. In addition, P13-5/A8 cells display very little, if any, surface expression of CD45, a receptor-linked tyrosine protein phosphatase, and lck, a lymphocyte-specific tyrosine kinase. On the other hand, they express a very high level of interleukin-2 receptor. A model is proposed that suggests that these cells are dependent on TPA because they have defects in both the PKC and tyrosine kinase signal transduction pathways, and that TPA compensates for these defects by providing a strong stimulus to the residual level of PKC. This variant may be useful for studying the interactions between tyrosine kinase and PKC pathways in controlling the various functions of T lymphocytes.
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PMID:Altered expression of protein kinase C, lck, and CD45 in a 12-O-tetradecanoylphorbol-13-acetate-dependent leukemic T-cell variant that expresses a high level of interleukin-2 receptor. 153 Aug 79

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.
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PMID:Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. 168 50

Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.
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PMID:Protein tyrosine phosphorylation and p56lck modification in IL-2 or phorbol ester-activated human natural killer cells. 169 99

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.
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PMID:Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL. 171 87

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.
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PMID:Multiple signal transduction pathways activated through the T cell receptor for antigen. 172 37

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.
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PMID:Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes. 204 Jun 25

Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.
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PMID:CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity. 216 57

Engagement of the TCR (CD3-Ti) by Ag/MHC, CD3 mAb, or lectin mitogen stimulates the very early tyrosine phosphorylation of several cellular substrates including TCR-zeta. The T cell specific protein-tyrosine kinase (PTK), p56lck, has been implicated in the tyrosine phosphorylation of TCR-zeta. However, the significance of this event with regard to CD3-Ti signal transduction remains unclear. Herein, we have investigated the effect of the selective PTK inhibitor genistein (4',5,7-trihydroxyisoflavone) on cellular events associated with activation via CD3-Ti triggering. Genistein inhibited the T cell PTK, p56lck, in a dose-dependent fashion with an ID50 = 40 microM. Genistein also inhibited CD3 mAb or PHA-induced TCR-zeta chain phosphorylation in intact peripheral blood T cells. Genistein blocked the expression of IL-2 and IL-2R (CD25) in T cells stimulated with PHA/PMA or CD3 mAb/PMA, but did not inhibit the de novo expression of the CD69 early activation Ag, which is induced primarily by a PKC-dependent pathway. IL-2 and CD25 expression induced by calcium ionophore A23187 and PMA was largely refractory to inhibition by genistein, suggesting an effect of the drug on calcium-dependent pathways stimulated via CD3-Ti triggering. In this last regard, genistein partially inhibited the CD3 mAb-induced rise in [Ca2+]i but did not inhibit PHA- or CD3 mAb-induced phosphatidylinositol hydrolysis. Consequently, protein-tyrosine phosphorylation does not appear to be a prerequisite for CD3-Ti-mediated activation of phosphatidylinositol-specific phospholipase C activity and PIP2 hydrolysis. An alternative role for PTK in CD3-Ti signal transduction is suggested.
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PMID:Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase. 217 80


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